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Background: Hypoxia is recognized as a key feature of cancer growth and is involved in various cellular processes, including proliferation, angiogenesis, and immune surveillance. Besides hypoxia-inducible factor 1-alpha (HIF-1α), which is the main mediator of hypoxia effects and can also be activated under normoxic conditions, little is known about its counterpart, HIF-2. This study focused on investigating the clinical and molecular landscape of HIF-2-altered urothelial carcinoma (UC). Methods: Publicly available next-generation sequencing (NGS) data from muscle-invasive UC cell lines and patient tumor samples from the MSK/TCGA 2020 cohort (n = 476) were interrogated for the level of expression (mRNA, protein) and presence of mutations, copy number variations, structural variants in the EPAS1 gene encoding HIF-2, and findings among various clinical (stage, grade, progression-free and overall survival) and molecular (tumor mutational burden, enriched gene expression) parameters were compared between altered and unaltered tumors. Results: 19% (7/37) of UC cell lines and 7% (27/380) of patients with muscle-invasive UC display high EPAS1 mRNA and protein expression or/and EPAS1 alterations. EPAS1-altered tumors are associated with higher stage, grade, and lymph node metastasis as well as with shorter PFS (14 vs. 51 months, q = 0.01) and OS (15 vs. 55 months, q = 0.01). EPAS1 mRNA expression is directly correlated with that of its target-genes, including VEGF, FLT1, KDR, DLL4, CDH5, ANGPT1 (q < 0.001). While there is a slightly higher tumor mutational burden in EPAS1-altered tumors (9.9 vs. 4.9 mut/Mb), they are enriched in and associated with genes promoting immune evasion, including ARID5B, SPINT1, AAK1, CLIC3, SORT1, SASH1, and FGFR3, respectively (q < 0.001). Conclusions: HIF-2-altered UC has an aggressive clinical and a distinct genomic and immunogenomic profile enriched in angiogenesis- and immune evasion-promoting genes.

Studying immune-mediated diseases (IMD) yields insights into the active immune system. In this thesis I cluster patients with IMD based on RNA-seq data using two extensions to clustering, and also cluster the diseases themselves using GWAS summary statistics. I conducted an extensive study of biclustering methods and found that a Bayesian biclustering method called SSLB had best performance on simulated datasets and recovered biologically relevant biclusters in a knockout mouse dataset and a sorted blood cell dataset. Through the study I developed tools for processing and analysing biclustering results. I applied this knowledge to perform biclustering, with SSLB, of a sorted blood cell dataset containing patients with six immune-mediated diseases. Amongst the biclusters was a bicluster recovering the genes that escape X-inactivation and a bicluster capturing type 1 interferon response, enriched for samples from patients with systemic lupus erythematosus. I found that whilst it is an advantage that biclustering has sufficient complexity to describe the immune system comprehensively, this also means that the results of the biclustering are themselves complex and thus interpretation can be a real challenge. In order to study RNA-seq data when summarised using key immune gene signatures, I developed DPMUnc, an extension to a Dirichlet process Bayesian clustering model which allows the uncertainty associated with the data points to be taken into account. I was then able to cluster patients based on their average gene expression across gene signatures, for example finding the expected enrichment of lupus patients in a cluster with high expression of interferon genes. I also clustered the immune-mediated diseases themselves using GWAS summary statistics. DPMUnc separated autoimmune from autoinflammatory diseases and isolated other subgroups such as the EGPA subtypes and multiple sclerosis. This thesis thus reveals an interesting trade-off between the complexity of the data and the utility of the results. Biclustering is sufficiently complex for the heterogeneity of the data but the results are difficult to interpret. In contrast, although some information is lost by summarising into key gene signatures, it allows for more concrete analysis.

The naked mole rat has unique biologic characteristics that include atypical inflammatory responses. Lipopolysaccharide induces inflammation which triggers brain centers controlling feeding, and behavior to result in “sick animal behavior”. We characterized the bodyweight, locomotor, and other behavioral responses of this rodent to lipopolysaccharide administration. Lipopolysaccharide caused weight losses, which were not prevented by TAK 242. In the open field test, lipopolysaccharide did not depress locomotion, while urination, defecation, and activity freezing were rare. The animals exhibited walling but not rearing and fast backward movements that were unaffected by lipopolysaccharide. Failure to depress locomotion suggests either a unique immunity-brain crosstalk or motor responses/centers that tolerate depressive effects of inflammation. The absence of activity freezing and rarity of urination and defecation suggests that novel environments or lipopolysaccharide do not induce anxiety, or that anxiety is expressed differently in the animal. The absence of rearing could be due to the design of the animal’s locomotor apparatus while fast backward movement could be a mechanism for quick escape from threats in the tunnels of their habitat. Our results elucidate the unique biology of this rodent, which elicits interest in the animal as a model for inflammatory research, although the findings require mechanistic corroborations.

A major challenge in the treatment of major depressive disorder (MDD) is relapse, which is defined as the return of depressive symptoms during a period of remission. Relapse rates in MDD are high, with approximately 50% of individuals relapsing following treatment of their first depressive episode, therefore early intervention to prevent relapse is crucial. Evidence suggests that immune dysregulation may be linked to longitudinal changes in depressive severity. However, it is currently unknown whether inflammation can predict future relapse in MDD. The objective of this project was to identify potential immune predictors of relapse in participants that responded to a treatment or a combination of treatments for MDD. A secondary objective was to investigate immune composition in efforts to stratify MDD individuals into more homogenous groups and further explore these groups in relation to clinical symptoms. This project is part of the Wellness Monitoring for Major Depressive Disorder longitudinal study (NCT02934334) of responders to antidepressant treatment conducted at 6 clinical sites across Canada. Montgomery Asberg Depression Rating Scale (MADRS) scores were used to assess depression severity and to categorize participants into ultrastable, unstable, and relapse groups. Plasma immune profiles were generated using the LEGENDplex Human Th Cytokine Panel immunoassay. Principal Component Analysis and Kruskal-Wallis tests of individual immune cytokines did not show differences between ultrastable, unstable, or relapse groups. Principal Component Analysis did reveal two cytokine clusters. Hierarchical Clustering analysis identified three distinct immune biotypes characterized by differing levels of Th cytokines and validated the presence of the cytokine clusters. Neither of these outcomes was predictive of relapse in this cohort. Our findings have shown that immune composition may serve as an important factor in parsing heterogeneity that is observed in this disorder through identification of distinct immune biotypes and highly interconnected cytokine subnetworks in major depression. The potential for immune biotypes for optimizing treatment regimens and relapse prevention necessitates further investigation and replication. Thesis Master of Science (MSc)

Depression is the most common mental disorder and has become a heavy burden in modern society. Clinical studies have identified early life stress as one of the high-risk factors for increased susceptibility to depression. Alteration of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress is one of the key risk factors for depression susceptibility related to early life stress. Laboratory animal studies have demonstrated that maternal separation (MS) for extended periods elicits HPA axis changes. These changes persist into adulthood and resemble those present in depressed adult individuals, including hyperactivity of the HPA axis. In addition, there is growing evidence that inflammation plays an important role in depression susceptibility concerned with early life stress. Individuals that have experienced MS have higher levels of pro-inflammatory cytokines and are susceptible to depression. Recently, it has been found that the gut microbiota plays an important role in regulating behavior and is also associated with depression. The translocation of gut microbiota and the change of gut microbiota composition caused by early stress may be a reason. In this review, we discussed the mechanisms by which early life stress contributes to the development of depression in terms of these factors. These studies have facilitated a systematic understanding of the pathogenesis of depression related to early life stress and will provide new ideas for the prevention and treatment of depression.

Purpose:This study aimed to determine the effects of an acute “train-low” nutritional protocol on markers of recovery optimization compared to standard recovery nutrition protocol.Methods:After completing a 2-hour high-intensity interval running protocol, 8 male endurance athletes consumed a standard dairy milk recovery beverage (CHO; 1.2 g/kg body mass [BM] of carbohydrate and 0.4 g/kg BM of protein) and a low-carbohydrate (L-CHO; isovolumetric with 0.35 g/kg BM of carbohydrate and 0.5 g/kg BM of protein) dairy milk beverage in a double-blind randomized crossover design. Venous blood and breath samples, nude BM, body water, and gastrointestinal symptom measurements were collected preexercise and during recovery. Muscle biopsy was performed at 0 hour and 2 hours of recovery. Participants returned to the laboratory the following morning to measure energy substrate oxidation and perform a 1-hour distance test.Results:The exercise protocol resulted in depletion of muscle glycogen stores (250 mmol/kg dry weight) and mild body-water losses (BM loss = 1.8%). Neither recovery beverage replenished muscle glycogen stores (279 mmol/kg dry weight) or prevented a decrease in bacterially stimulated neutrophil function (−21%). Both recovery beverages increased phosphorylation of mTORSer2448(main effect of time = P Ser9/total-GSK-3β occurred on CHO (P = .012). Blood glucose (P = .005) and insulin (P = .012) responses were significantly greater on CHO (618 mmol/L per 2 h and 3507 μIU/mL per 2 h, respectively) compared to L-CHO (559 mmol/L per 2 h and 1147 μIU/mL per 2 h, respectively). Rates of total fat oxidation were greater on CHO, but performance was not affected.Conclusion:A lower-carbohydrate recovery beverage consumed after exercise in a “train-low” nutritional protocol does not negatively impact recovery optimization outcomes.

Inflammation plays a significant role in the occurrence and development of multiple diseases. This study comprehensively reviews and presents literature from the last five years, showing that acupuncture indeed exerts strong anti-inflammatory effects in multiple biological systems, namely, the immune, digestive, respiratory, nervous, locomotory, circulatory, endocrine, and genitourinary systems. It is well known that localized acupuncture-mediated anti-inflammatory effects involve the regulation of multiple populations and functions of immune cells, including macrophages, granulocytes, mast cells, and T cells. In acupuncture stimulation, macrophages transform from the M1 to the M2 phenotype and the negative TLR4 regulator PPARγ is activated to inhibit the intracellular TLR/MyD88 and NOD signaling pathways. The downstream IκBα/NF-κB and P38 MAPK pathways are subsequently inhibited by acupuncture, followed by suppressed production of inflammasome and proinflammatory mediators. Acupuncture also modulates the balance of helper T cell populations. Furthermore, it inhibits oxidative stress by enhancing SOD activity via the Nrf2/HO-1 pathway and eliminates the generation of oxygen free radicals, thereby preventing inflammatory cell infiltration. The anti-inflammatory effects of acupuncture on different biological systems are also specific to individual organ microenvironments. As part of its anti-inflammatory action, acupuncture deforms connective tissue and upregulates the secretion of various molecules in acupoints, further activating the NF-κB, MAPK, and ERK pathways in mast cells, fibroblasts, keratinocytes, and monocytes/macrophages. The somatic afferents present in acupuncture-activated acupoints also convey sensory signals to the spinal cord, brainstem, and hypothalamic neurons. Upon information integration in the brain, acupuncture further stimulates multiple neuro-immune pathways, including the cholinergic anti-inflammatory, vagus-adrenal medulla-dopamine, and sympathetic pathways, as well as the hypothalamus-pituitary-adrenal axis, ultimately acting immune cells via the release of crucial neurotransmitters and hormones. This review provides a scientific and reliable basis and viewpoints for the clinical application of acupuncture in various inflammatory conditions.

Yucheng Fu, Guoyu He, Zhuochao Liu, Jun Wang, Zhusheng Zhang, Qiyuan Bao, Junxiang Wen, Zhijian Jin, Weibin Zhang Department of Orthopedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopedics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, People’s Republic of ChinaCorrespondence: Weibin ZhangDepartment of Orthopedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopedics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin Er Road, Shanghai, 200025, People’s Republic of ChinaTel +86-21-64370045, Ext 666983Fax +86-21-54660217Email zhangweibin10368@163.comBackground: Inflammatory response took part in the progression of tumor and was regarded as the hallmark of cancer. However, the prognostic relationship between osteosarcoma and inflammatory response-associated genes (IRGs) was unclear. This research aimed to explore the correlations between osteosarcoma prognosis and IRG signature.Methods: The inflammatory response-associated differentially expressed messenger RNAs (DEmRNAs) were screened out through Gene Expression Omnibus (GEO) and Molecular Signature Database (MSigDB) databases. Univariate and multivariate cox regression analyses were utilized to construct the IRG signature. The prognostic value of signature was investigated through Kaplan–Meier (KM) survival curve and nomogram. DEmRNAs among high and low inflammatory response-associated risks were identified and functional enrichment analyses were conducted. ESTIMATE, CIBERSORT and single-sample gene set enrichment analyses (ssGSEA) were implied to reveal the alterations in immune infiltration. All the above results were validated in Target database. The expression of IRGs was also validated in different cell lines by quantitative real-time PCR (qRT-PCR) and osteosarcoma patient samples by immunohistochemistry.Results: The IRG signature that consisted of two genes (MYC, CLEC5A) was established. In training and validation datasets, patients with lower risk scores survived longer and the IRG signature was confirmed as the independent prognostic factor in osteosarcoma. The nomogram was constructed and the calibration curves demonstrated the reliability of this model. Functional analysis of risk score-associated DEmRNAs indicated that immune-related pathways and functions were significantly enriched. ssGSEA revealed that 14 immune cells and 11 immune functions were significantly dysregulated. The qRT-PCR results indicated IRGs were significantly differently expressed in osteosarcoma and osteoblast cell lines. The immunohistochemistry analyses of patients’ samples revealed the same result.Conclusion: The novel osteosarcoma inflammatory response-associated prognostic signature was established and validated in this study. This model could serve as the biomarker and therapeutic target for osteosarcoma in the future.Keywords: osteosarcoma, inflammatory response, metastasis, prognosis, immune

Background Inflammation and increases in inflammatory cytokines are common findings in psychiatric disorders such as schizophrenia (SCZ), bipolar disorder (BD), and major depressive disorder (MDD). Meta-analyses of studies that measured circulating cytokines have provided evidence of innate inflammation across all three disorders, with some overlap of inflammatory cytokines such as IL-6 and TNF-α. However, differences across disorders were also identified, including increased IL-4 in BD that suggest different immune mechanisms may be involved depending on the type of disorder present. Methods We sought to identify if the presence or absence of an affective disorder in first-episode psychotic (FEP) patients was associated with variations in cytokine production after stimulation of peripheral blood mononuclear cells (PBMC). 98 participants were recruited and grouped into healthy controls (n = 45) and first-episode psychosis patients (n = 53). Psychosis patients were further grouped by presence (AFF; n = 22) or lack (NON; n = 31) of an affective disorder. We cultured isolated PBMC from all participants for 48 h at 37 °C under four separate conditions; (1) culture media alone for baseline, or the following three stimulatory conditions: (2) 25 ng/mL lipopolysaccharide (LPS), (3) 10 ng/mL phytohemagglutinin (PHA), and (4) 125 ng/ml α-CD3 plus 250 ng/ml α-CD28. Supernatants collected at 48 h were analyzed using multiplex Luminex assay to identify differences in cytokine and chemokine production. Results from these assays were then correlated to patient clinical assessments for positive and negative symptoms common to psychotic disorders. Results We found that PBMC from affective FEP patients produced higher concentrations of cytokines associated with both innate and adaptive immunity after stimulation than non-affective FEP patients and healthy controls. More specifically, the AFF PBMC produced increased tumor necrosis fctor (TNF)-α, interleukin (IL)-1β, IL-6, and others associated with innate inflammation. PBMC from AFF also produced increased IL-4, IL-17, interferon (IFN)γ, and other cytokines associated with adaptive immune activation, depending on stimulation. Additionally, inflammatory cytokines that differed at rest and after LPS stimulation correlated with Scale for the Assessment of Negative Symptoms (SANS) scores. Conclusions Our findings suggest that immune dysfunction in affective psychosis may differ from that of primary psychotic disorders, and inflammation may be associated with increased negative symptoms. These findings could be helpful in determining clinical diagnosis after first psychotic episode.

Background and Objectives: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a multifactorial disease resulting from inflammation of the nasal cavity and paranasal sinuses. Systemic allergic inflammation is an important cause of CRSwNP; however, the effect of local allergic inflammation is unclear. This study was designed to investigate the effect of local allergic inflammation in CRSwNP.Materials and Methods: The study included 11 patients with CRSwNP and 18 control subjects. Olfactory function was measured with the Korean Version of Sniffin’s stick test. Nasal lavage fluids (NLFs) were collected from all subjects and analyzed for total IgE, eosinophilic cationic protein (ECP), and cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-4, IL-10, IL-17A, interferon-γ). Flow cytometry was used to measure various inflammatory cells in the NAL fluids.Results: On analysis of flow cytometry and enzyme-linked immunosorbent assay, we found that CRSwNP patients had significantly increased eosinophil (%) and ECP levels in NLFs. In addition, there was significant local-systemic correlation between ECP level in NLFs and blood eosinophils (%) (r=0.391); however, there was no significant association between eosinophils (%) in NLFs and blood eosinophils. Moreover, in CRSwNP patients, the severity of disease was related with blood eosinophil (%), eosinophil (%), and ECP levels in NLFs, whereas olfactory function was associated with blood eosinophil (%) and ECP levels in NLFs.Conclusion: CRSwNP is a disease with high allergic inflammation that has negative impacts on the severity of disease and olfactory function. Therefore, we suggest that control of local allergic inflammation will be helpful to treat CRSwNP patients.

Despite increasingly thorough mechanistic understanding of the dominant genetic drivers of gastrointestinal (GI) tumorigenesis (e.g., Ras/Raf, TP53, etc.), only a small proportion of these molecular alterations are therapeutically actionable. In an attempt to address this therapeutic impasse, our group has proposed an innovative extreme outlier model to identify novel cooperative molecular vulnerabilities in high-risk GI cancers which dictate prognosis, correlate with distinct patterns of metastasis, and define therapeutic sensitivity or resistance. Our model also proposes comprehensive investigation of their downstream transcriptomic, immunomic, metabolic, or upstream epigenomic cellular consequences to reveal novel therapeutic targets in previously “undruggable” tumors with high-risk genomic features. Leveraging this methodology, our and others’ data reveal that the genomic cooperativity between Ras and p53 alterations is not only prognostically relevant in GI malignancy, but may also represent the incipient molecular events that initiate and sustain innate immunoregulatory signaling networks within the GI tumor microenvironment, driving T-cell exclusion and therapeutic resistance in these cancers. As such, deciphering the unique transcriptional programs encoded by Ras-p53 cooperativity that promote innate immune trafficking and chronic inflammatory tumor-stromal-immune crosstalk may uncover immunologic vulnerabilities that could be exploited to develop novel therapeutic strategies for these difficult-to-treat malignancies.

In recent years, an increasing number of studies have reported that intestinal microbiota have an important effect on tumour immunity by affecting the tumour microenvironment (TME). The intestinal microbiota are closely associated with various immune cells, such as T lymphocytes, natural killer cells (NK cells) and macrophages. Some bacteria, such as Akkermansia muciniphila (A. muciniphila) and Lactobacillus reuteri (L. reuteri), have been shown to improve the effect of tumour immunity. Furthermore, microbial imbalance, such as the increased abundance of Fusobacterium nucleatum (F. nucleatum) and Helicobacter hepaticus (H. hepaticus), generally causes tumour formation and progression. In addition, some microbiota also play important roles in tumour immunotherapy, especially PD‐L1‐related therapies. Therefore, what is the relationship between these processes and how do they affect each other? In this review, we summarize the interactions and corresponding mechanisms among the intestinal microbiota, immune system and TME to facilitate the research and development of new targeted drugs and provide new approaches to tumour therapy., We systematically explored the relationship among intestinal microbiota, immunity and tumour microenvironment. Besides, we also discussed the combined effect of gut microbiota and ICB. In short, the potential of gut microbiota in tumour therapy is promising.

Emerging data indicates that non-coding RNAs (ncRNAs) represent more than just “junk sequences” of the genome and have been found to be involved in multiple diseases by regulating various biological process, including the activation of inflammasomes. As an important aspect of innate immunity, inflammasomes are large immune multiprotein complexes that tightly regulate the production of pro-inflammatory cytokines and mediate pyroptosis; the activation of the inflammasomes is a vital biological process in inflammatory diseases. Recent studies have emphasized the function of ncRNAs in the fine control of inflammasomes activation either by directly targeting components of the inflammasomes or by controlling the activity of various factors that control the activation of inflammasomes; consequently, ncRNAs may represent potential therapeutic targets for inflammatory diseases. Understanding the precise role of ncRNAs in controlling the activation of inflammasomes will help us to design targeted therapies for multiple inflammatory diseases. In this review, we summarize the regulatory role and therapeutic potential of ncRNAs in the activation of inflammasomes by focusing on a range of inflammatory diseases, including microbial infection, sterile inflammatory diseases, and fibrosis-related diseases. Our goal is to provide new ideas and perspectives for future research.

Xiaoxiao Liu,1 Xiaobo Shi,1 Wei Guo,1 Yue Ke,1 Yuxing Li,1 Shupei Pan,1 Xiaona Li,2 Mei Liu,3 Mingzhu Liu,4 Yuchen Wang,1 Qinli Ruan,1 Hongbing Ma1 1Department of Radiation Oncology, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710004, People’s Republic of China; 2Department of Dermatology, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710004, People’s Republic of China; 3Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710004, People’s Republic of China; 4Department of Gastroenterology, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710004, People’s Republic of ChinaCorrespondence: Hongbing MaDepartment of Radiation Oncology, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710004, People’s Republic of ChinaTel +86 13991845066Email mhbxian@126.comPurpose: Ferroptosis and long non-coding RNA (lncRNA) expression signatures have been associated with the clinical progression and immune-contexture of different solid tumors. The study aimed to identify a prognostic signature of ferroptosis-related lncRNAs (falncRNAs) to forecast the immune scenery and immunotherapy response in esophageal cancer (EC).Patients and Methods: Gene expression profiles of EC were extracted from The Cancer Genome Atlas (TCGA) database, and ferroptosis-related genes were downloaded from the FerrDb database, which identified differentially expressed falncRNAs (DEfalncRNAs) via differential analysis. DEfalncRNA pairs associated with prognosis were identified by constructing a matrix, univariate and least absolute shrinkage and selection operator (LASSO) analysis. The prognostic signature was constructed by multivariate analysis. We appraised the forecasting capability of prognostic signature in survival, clinicopathological features, immune landscape, efficacy of immunotherapy, and drug sensitivity. The potential molecular mechanism of signature was investigated by gene set enrichment analysis (GSEA).Results: We obtained 18 DEfalncRNA pairs to define a novel prognostic signature that was determined on a discovery cohort of 158 tumor samples and 11 adjacent normal tissues from TCGA and internally validated, with the definition of high- vs low-risk groups based on 3 years overall survival. We demonstrated that the high- vs low-risk groups differed for clinical parameters and computationally predicted drug sensitivity and tumor immune contexture, with the high-risk group having worse survival, more aggressive disease (node involvement, metastasis), reduced drug sensitivity, higher tumor mutation load, and gene signatures of infiltration of pro-tumoral immune cell subsets. The GSEA results revealed that ferroptosis and immunoregulatory pathways were significantly enriched in the high-risk group.Conclusion: The prognostic signature based on falncRNAs has the potential to forecast the survival, immune scenery, efficacy of immunotherapy, and drug sensitivity of EC, which is helpful for clinical prediction and individualized treatment.Keywords: esophageal cancer, ferroptosis, lncRNA, immune, signature, prognosis

Background C-X-C chemokine receptor 4 (CXCR4) is a specific receptor of stromal cell-derived factor-1, also known as CXCL12. The interaction between CXCL12 and its receptor CXCR4 can activate various signaling pathways, including gene expression, cell proliferation, migration, tumorigenesis, angiogenesis, etc. Although there is evidence to support the association between CXCR4 and some cancers, there is no pan-cancer analysis. To fill this gap, we analyzed the role of CXCR4 in cancer-based on The Cancer Genome Atlas (TCGA). Methods We used TCGA, Genotype-Tissue Expression (GTEx) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases to analyze the expression, variation and phosphorylation of CXCR4 in different cancers. At the same time, we also carried out Kyoto Encyclopedia of Genes (KEGG) and Gene Ontology (GO) enrichment analysis. Results We found that CXCR4 expression was significantly increased in bladder urothelial carcinoma (BLCA) and other cancers, and CXCR4 expression in BLCA, cervical squamous cell carcinoma (CESC) and other cancers was related to tumor stage. CXCR4 expression was positively correlated with tumor-associated fibroblasts in BLCA, breast adenocarcinoma (BRCA), CESC and other cancers. GO analysis showed that CXCR4-related genes were mainly enriched in biological processes (BPs) and cellular components (CCs). KEGG analysis showed that CXCR4 was mainly involved in “chemokine signaling pathway”, “natural killer cell-mediated cytotoxicity”, and “JAK-STAT signaling pathway”. Conclusions The expression of CXCR4 in different cancers has different effects on the prognosis of patients and the infiltration of immune cells.

Chronic widespread pain (CWP), including fibromyalgia (FM), is characterized by generalized musculoskeletal pain and hyperalgesia. Plasma proteins from proteomics (non-targeted) and from targeted inflammatory panels (cytokines/chemokines) differentiate CWP/FM from controls. The importance of proteins obtained from these two sources, the protein-protein association network, and the biological processes involved were investigated. Plasma proteins from women with CWP (n = 15) and CON (n = 23) were analyzed using two-dimensional gel electrophoresis analysis and a multiplex proximity extension assay for analysis of cytokines/chemokines. Associations between the proteins and group were multivarietly analyzed. The protein-protein association network and the biological processes according to the Gene Ontology were investigated. Proteins from both sources were important for group differentiation; the majority from the two-dimensional gel electrophoresis analysis. 58 proteins significantly differentiated the two groups (R-2 = 0.83). A significantly enriched network was found; biological processes were acute phase response, complement activation, and innate immune response. As with other studies, this study shows that plasma proteins can differentiate CWP from healthy subjects. Focusing on cytokines/chemokines is not sufficient to grasp the peripheral biological processes that maintain CWP/FM since our results show that other components of the immune and inflammation systems are also highly significant.

A teleost’s kidney was divided into head kidney and trunk kidney. The head kidney is an important lymphatic organ, while the trunk kidney mainly performs osmotic pressure regulation and excretion functions. Previous studies have shown that the teleost’s head kidney exerts a strong immune response against pathogen invasion, while the mechanism of immune response in the trunk kidney is still rarely reported. Therefore, in this study, we established an Infectious hematopoietic necrosis virus (IHNV) immersion infection model to compare the similarities and differences of immune response mechanisms between the head kidney and trunk kidney against viral infection. The results showed that IHNV infection causes severe tissue damage and inflammatory reaction in the head and trunk kidney, triggers a series of interferon cascade reactions, and produces strong immune response. In addition, the transcriptome data showed that the head kidney and trunk kidney had similar immune response mechanisms, which showed that the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway were activated. In conclusion, despite functional differentiation, the teleost’s trunk kidney still has a strong immune response, especially the interferon-stimulated genes, which have stronger immune response in the trunk kidney than in the head kidney when responding to IHNV infection. This study contributes to a more comprehensive understanding of the teleost immune system and enriches the theory of kidney immunity in teleosts.

We investigated antibody and coronavirus disease 2019 (COVID-19)-specific T-cell mediated responses via ultra-deep immunosequencing of the T-cell receptor (TCR) repertoire in patients with plasma cell dyscrasias (PCD). We identified 364 patients with PCD who underwent spike antibody testing using commercially available spike-receptor binding domain immunoglobulin G antibodies ≥2 weeks after completion of the initial two doses of mRNA vaccines or one dose of JNJ-78436735. A total of 56 patients underwent TCR immunosequencing after vaccination. Overall, 86% tested within 6 months of vaccination had detectable spike antibodies. Increasing age, use of anti-CD38 or anti-B-cell maturation antigen therapy, and receipt of BNT162b2 (vs. mRNA-1273) were associated with lower antibody titres. We observed an increased proportion of TCRs associated with surface glycoprotein regions of the COVID-19 genome after vaccination, consistent with spike-specific T-cell responses. The median spike-specific T-cell breadth was 3.11 × 10-5 , comparable to those in healthy populations after vaccination. Although spike-specific T-cell breadth correlated with antibody titres, patients without antibody responses also demonstrated spike-specific T-cell responses. Patients receiving mRNA-1273 had higher median spike-specific T-cell breadth than those receiving BNT162b2 (p=0.01). Although patients with PCD are often immunocompromised due to underlying disease and treatments, COVID-19 vaccination can still elicit humoral and T-cell responses and remain an important intervention in this patient population.

Altitude is the main external environmental pressure affecting the production performance of Tibetan sheep, and the adaptive evolution of many years has formed a certain response mechanism. However, there are few reports on the response of ruminal microbiota and host genomes of Tibetan sheep to high-altitude environments. Here, we conducted an integrated analysis of volatile fatty acids (VFAs), microbial diversity (16S rRNA), epithelial morphology, and epithelial transcriptome in the rumen of Tibetan sheep at different altitudes to understand the changes in ruminal microbiota–host interaction in response to high altitude. The differences in the nutritional quality of forage at different altitudes, especially the differences in fiber content (ADF/NDF), led to changes in rumen VFAs of Tibetan sheep, in which the A/P value (acetic acid/propionic acid) was significantly decreased (p < 0.05). In addition, the concentrations of IgA and IgG in Middle-altitude (MA) and High-altitude Tibetan sheep (HA) were significantly increased (p < 0.05), while the concentrations of IgM were significantly increased in MA (p < 0.05). Morphological results showed that the width of the rumen papilla and the thickness of the basal layer increased significantly in HA Tibetan sheep (p < 0.05). The 16S rRNA analysis found that the rumen microbial diversity of Tibetan sheep gradually decreased with increasing altitude, and there were some differences in phylum- and genus-level microbes at the three altitudes. RDA analysis found that the abundance of the Rikenellaceae RC9 gut group and the Ruminococcaceae NK4A214 group increased with altitudes. Furthermore, a functional analysis of the KEGG microbial database found the “lipid metabolism” function of HA Tibetan sheep to be significantly enriched. WGCNA revealed that five gene modules were enriched in “energy production and conversion”, “lipid transport and metabolism”, and “defense mechanisms”, and cooperated with microbiota to regulate rumen fermentation and epithelial immune barrier function, so as to improve the metabolism and immune level of Tibetan sheep at high altitude.

Background: Hepatocellular carcinoma (HCC) originates from the hepatocytes and accounts for 90% of liver cancer. The study intends to identify novel prognostic biomarkers for predicting the prognosis of HCC patients based on TCGA and GSE14520 cohorts. Methods: Differential analysis was employed to obtain the DEGs (Differentially Expressed Genes) of the TCGA-LIHC-TPM cohort. The lasso regression analysis was applied to build the prognosis model through using the TCGA cohort as the training group and the GSE14520 cohort as the testing group. Next, based on the prognosis model, we performed the following analyses: the survival analysis, the independent prognosis analysis, the clinical feature analysis, the mutation analysis, the immune cell infiltration analysis, the tumor microenvironment analysis, and the drug sensitivity analysis. Finally, the survival time of HCC patients was predicted by constructing nomograms. Results: Through the lasso regression analysis, we obtained a prognosis model of ten genes including BIRC5 (baculoviral IAP repeat containing 5), CDK4 (cyclin-dependent kinase 4), DCK (deoxycytidine kinase), HSPA4 (heat shock protein family A member 4), HSP90AA1 (heat shock protein 90 α family class A member 1), PSMD2 (Proteasome 26S Subunit Ubiquitin Receptor, Non-ATPase 2), IL1RN (interleukin 1 receptor antagonist), PGF (placental growth factor), SPP1 (secreted phosphoprotein 1), and STC2 (stanniocalcin 2). First, we found that the risk score is an independent prognosis factor and is related to the clinical features of HCC patients, covering AFP (α-fetoprotein) and stage. Second, we observed that the p53 mutation was the most obvious mutation between the high-risk and low-risk groups. Third, we also discovered that the risk score is related to some immune cells, covering B cells, T cells, dendritic, macrophages, neutrophils, etc. Fourth, the high-risk group possesses a lower TIDE score, a higher expression of immune checkpoints, and higher ESTIMATE score. Finally, nomograms include the clinical features and risk signatures, displaying the clinical utility of the signature in the survival prediction of HCC patients. Conclusions: Through the comprehensive analysis, we constructed an immune-related prognosis model to predict the survival of HCC patients. In addition to predicting the survival time of HCC patients, this model significantly correlates with the tumor microenvironment. Furthermore, we concluded that these ten immune-related genes (BIRC5, CDK4, DCK, HSPA4, HSP90AA1, PSMD2, IL1RN, PGF, SPP1, and STC2) serve as novel targets for antitumor immunity. Therefore, this study plays a significant role in exploring the clinical application of immune-related genes.

Xiaoning Cen,1,&ast; Yunmei Huang,2,&ast; Zhuangnian Lu,3,&ast; Wenjun Shao,4 Chenyi Zhuo,1 Chongchan Bao,5 Shi Feng,5 Cheng Wei,5 Xiqiang Tang,5 Lijun Cen,5 Wenwen Guo,5 Xinru Tian,5 Qianli Tang,5 Xusen Huang1 1General Surgery, Affiliated Hospital of YouJiang Medical University for Nationalities, Baise, 533000, Guangxi, People’s Republic of China; 2Department of Pathology, Affiliated Hospital of YouJiang Medical University for Nationalities, Baise, 533000, Guangxi, People’s Republic of China; 3Department of Pediatrics, Affiliated Hospital of YouJiang Medical University for Nationalities, Baise, 533000, Guangxi, People’s Republic of China; 4Medical college of Soochow University, Suzhou, Jiangsu, 215000, People’s Republic of China; 5YouJiang Medical University for Nationalities, Baise, 533000, Guangxi, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Qianli TangYouJiang Medical University for Nationalities, Baise, 533000, Guangxi, People’s Republic of ChinaEmail htmgx@163.comXusen HuangGeneral Surgery, Affiliated Hospital of YouJiang Medical University for Nationalities, Baise, 533000, Guangxi, People’s Republic of ChinaEmail hxsfy@163.comBackground: LncRNAs play an important role in tumor initiation and development. However, the underlying involvement of lncRNA expression in colorectal carcinoma remains to be clarified.Methods: All analyses were performed in R software v4.0, SPSS v13.0, and GraphPad Prism 8. The “limma” package was used to identify differentially expressed lncRNAs between two groups with the threshold of |logFC| > 1 and P < 0.05. The “Survival” package was used to conduct survival analysis. HCT8 and SE480 cell lines were used to conduct further phenotype experiments, including transwell, wound-healing, CCK8 and colony formation assay. Gene set enrichment analysis was used to explore the biological pathway difference in high and low IGFL2-AS1 patients.Results: The lncRNA IGFL2-AS1 was highly expressed in colon adenocarcinoma (COAD) tissue and cell lines (HCT116, HCT8, HCT129, and SW480). The COAD patients with high IGFL2-AS1 were associated with a worse prognosis. Meanwhile, the knockdown of IGFL2-AS1 could significantly suppress the proliferation and invasion of COAD cells. Gene set enrichment analysis showed that the top five biological pathways involving IGFL2-AS1 were angiogenesis, epithelial–mesenchymal transition, KRAS signaling, myogenesis, and coagulation. Western blot results showed that the inhibition of IGFL2-AS1 could significantly reduce the N-cadherin, HIF1A and KRAS protein expression, yet increase the E-cadherin protein level. IGFL2-AS1 was also positively correlated with M0 macrophages, M2 macrophages, and neutrophils but negatively correlated with CD4+ memory T cells and CD8+ T cells.Conclusion: IGFL1-AS1 could seriously worsen patient outcomes and facilitate COAD progression, thus serving as an independent tumor marker.Keywords: IGFL2-AS1, lncRNA, colon cancer, immune

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by SARS-CoV-2, a newly discovered coronavirus that exhibits many similarities with the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (SARS-CoV and MERS-CoV, respectively). The definite pathogenesis and immunological influences of SARS-CoV-2 have not been fully elucidated. Therefore, we constructed a brief summary comparison of SARS-CoV-2, SARS-CoV, and MERS-CoV infections regarding their immunological changes. In addition, we further investigated the immunological differences between severe and nonsevere COVID-19 cases, and we searched for possible immunological predictors of the patient outcome by reviewing case series studies to date. Possible immunological predictors of a poor outcome are leukocytosis, neutrophilia, lymphopenia (both CD4 and CD8 T cells), an increased neutrophil-to-lymphocyte ratio (NLR), and increased levels of pro-inflammatory cytokines (IL-6 and TNF-α), Th1 cytokines (IL-2 and IFN-γ), regulatory T cell cytokines (IL-10) and Th17 cytokines (IL-17). A more precise immunological map needs to be established, which may assist in diagnosing this disease and facilitate immunological precision medicine treatment.

Objective To investigate the molecular characteristics in tumor immune microenvironment that affect long-term survival of patients with pancreatic adenocarcinoma (PAAD). Methods The tumor related genetic features of a female PAAD patient (over 13-year survival) who suffered from multiple recurrences and metastases, and six operations over one decade were investigated deeply. Genomic features and immune microenvironment signatures of her primary lesion as well as six metastatic tumors at different time-points were characterized. Results High-frequency clonal neoantigenic mutations identified in these specimens revealed the significant associations between clonal neoantigens with her prognosis after each surgery. Meanwhile, the TCGA and ICGC databases were employed to analyse the function of KRAS G12V in pancreatic cancer. Conclusions The genomic analysis of clonal neoantigens combined with tumor immune microenvironment could promote the understandings of personalized prognostic evaluation and the stratification of resected PAAD individuals with better outcome.

Natural food and beverage processing is the result of natural food processing sourced from family plants which are usually grown in the yard around the house. Natural processed food and beverage products are not only nutritious and nutritional enhancer but also as cooking spices that contain high antioxidants. Utilization of natural processed food and beverage products as an effort to prevent disease, health care during public health emergencies and especially during the national disaster for the corona virus disease 2019 (COVID-19). The socialization of natural processed food and beverage products is carried out in three stages. The first stage is to prepare and select the types of medicinal plants that are often planted by the Petoaha community around the house. The second stage is material education about the benefits of family medicinal plants (TOGA), processed natural food and beverage products, as an effort to increase immunity in the midst of the COVID-19 pandemic. While the last stage is to provide an explanation of how to process food and beverage ingredients and to clarify the use of natural food and beverage processed products as health care, increase immunity and disease prevention during the COVID-19 pandemic. The result of this activity is to increase the knowledge, insight and awareness of the Petoaha community about the importance of planting TOGA plants and their processed food or beverage products to increase immunity during the Covid 19 pandemic.. Abstrak Pengolahan makanan dan minuman alami merupakan hasil olahan pangan alami yang bersumber dari tanaman keluarga yang biasanya ditanam di pekarangan sekitar rumah. Produk makanan dan minuman olahan alami tidak hanya berkhasiat dan penambah gizi tetapi juga sebagai bumbu masak yang mengandung antioksidan yang tinggi. Pemanfaatan produk olahan makanan dan minuman alami sebagai upaya pencegahan penyakit, pemeliharaan kesehatan pada saat keadaan darurat kesehatan masyarakat dan khususnya pada saat bencana nasional penyakit virus corona 2019 (COVID 19). Sosialisasi produk olahan makanan dan minuman alami dilakukan dalam tiga tahap. Tahap pertama yaitu menyiapkan dan menyeleksi jenis tanaman obat yang sering ditanam masyarakat Petoaha di sekitar rumah. Tahap kedua adalah edukasi materi tentang manfaat tanaman obat keluarga (TOGA), olahan produk makanan dan minuman alamiah, sebagai upaya peningkatan imunitas di tengah pandemi COVID-19. Sedangkan tahap terakhir yaitu memberikan penjelasan tentang cara mengolah bahan makanan dan minuman serta memperjelas penggunaan produk olahan makanan dan minuman alami sebagai perawatan kesehatan, peningkatan immun dan pencegahan penyakit selama pandemi covid 19. Hasil dari kegiatan ini adalah untuk meningkatkan pengetahuan, wawasan dan kesadaran masyarakat Petoaha tentang pentingnya menanam tanaman TOGA dan produk olahan makanan atau minumannya guna meningkatkan imun pada saat pandemi Covid 19.

Qinke Li, Maoqing Lu, Zhechuan Zhang, Ronggui Zhang Department of Urology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of ChinaCorrespondence: Ronggui ZhangDepartment of Urology, The Second Affiliated Hospital, Linjiang Road, Chongqing Medical University, Chongqing, 400010, People’s Republic of ChinaTel +86-23-13983790901Fax +23-63832133Email zrgcqmu@126.comBackground: Heat shock proteins (HSPs) are widely involved in tumor occurrence and development and are prognostic markers for multiple tumors. However, the role of HSPs in clear cell renal cell carcinoma (ccRCC) remains unclear.Methods: We used Cytoscape to identify hub genes in the ccRCC single-cell sequencing data set from the Gene Expression Omnibus (GEO) data repository. We identified subtypes, C1 and C2, of The Cancer Genome Atlas (TCGA) patients based on the expression of hub genes using unsupervised consensus clustering. Principal component analysis (PCA) was used to verify the clustering differences, and Kaplan–Meier (K-M) estimate was used to verify the survival differences between C1 and C2 patients. We used TIMER 2.0 and CIBERSORT to evaluate the immune cell infiltration of HSP genes and C1 and C2 patients. The R package “pRRophetic” was used to evaluate the sensitivity in C1 and C2 patients to the four first-line treatment drugs.Results: We identified six hub genes (HSP90AA1, HSPH1, HSPA1B, HSPA8, and HSPA1A) encoding HSP, five of which were significantly downregulated in TCGA group, and four had a protective effect on prognosis (p < 0.05). Survival analysis showed that C1 patients had a better overall survival (p < 0.001). TIMER 2.0 analysis showed that three HSP genes were significantly correlated with the infiltration of CD4+ T cells and CD4+ Th1 cells (|cor|> 0.5, p< 0.001). CIBERSORT showed significant differences in multiple infiltrating immune cells between C1 and C2 patients. Meanwhile, the expression of PD1 was significantly lower in C1 patients than in C2 patients, and the expression of PDL1 is the another way around. Drug sensitivity analysis showed that C1 patients were more sensitive to sorafenib, pazopanib, and axitinib (p < 0.001).Conclusion: Our research revealed two molecular subtypes of ccRCC based on 6 HSP genes, and revealed significant differences between the two subtypes in terms of clinical prognosis, immune infiltration, and drug sensitivity.Keywords: ccRCC, bioinformatics, immune, molecular subtypes, prognosis

Introduction Disseminated herpes zoster (DHZ) is a complication of herpes zoster (HZ) associated with an immunocompromised state. Immunocompromised conditions in systemic lupus erythematosus (SLE) and lupus nephritis increase the incidence of DHZ. Case A 14-year-old girl was consulted with a complaint of painful reddish blisters for the previous 4 days. At first, blisters only appeared on the right leg, then multiplied and spread throughout the body. The patient had been diagnosed with SLE since 3 months and 2 months later also diagnosed with lupus nephritis. The patient received methylprednisolone pulse dose 1 g for 3 days, then maintenance dose 1×80 mg every 2 days for 2 weeks before blisters appeared. Dermatological examination of the facial region, anterior and posterior trunk, gluteus, superior limb dextra and sinistra, dorsum manus, cruris dextra, femur dextra lateral and plantar pedis presented vesicles with an erythematous base, and some other confluent vesicles became bullae, well defined, with an irregular border, varied in size and shape. An investigation by Tzanck smears in bullae and vesicles showed polymorphonuclear cells (PMN) and multinucleated giant cells (MGC). Laboratory tests showed anemia, neutrophilia, and lymphopenia. The patient was diagnosed as HZD. The patient received oral acyclovir 5×800mg for 10 days and obtained clinical improvement. Conclusion Although the onset of HZ in children is less common, it may occur in children with SLE comorbidity. Complications of DHZ in SLE patients may be due to immunological abnormalities, lymphopenia conditions, and immunosuppression therapy with high-dose glucocorticoids (GC). In addition, the incidence of both HZ and DHZ increases in lupus nephritis patients compared with SLE.

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of various chemotherapeutic agents, including oxaliplatin. It is highly prevalent amongst cancer patients, causing sensory abnormalities and pain. Unfortunately, as the underlying mechanisms remain poorly understood, effective therapeutics are lacking. Neuro-immune interactions have been highlighted as potential contributors to the development and maintenance of CIPN, however, whether this is the case in oxaliplatin-induced peripheral neuropathy (OIPN) is yet to be fully established. Methods: In this study we used flow cytometry to examine the peripheral immune response of male C57BL/6 mice following both single and repeated oxaliplatin administration. In animals exposed to repeated dosing, we also undertook mechanical and thermal behavioural assays to investigate how oxaliplatin alters phenotype, and conducted RT-qPCR experiments on bone marrow derived macrophages in order to further inspect the effects of oxaliplatin on immune cells. Results: In contrast to other reports, we failed to observe substantial changes in overall leukocyte, lymphocyte or myeloid cell numbers in dorsal root ganglia, sciatic nerves or inguinal lymph nodes. We did however note subtle, tissue-dependant alterations in several myeloid subpopulations following repeated dosing. These included a significant reduction in MHCII antigen presenting cells in the sciatic nerve and an increase in infiltrating cell types into the inguinal lymph nodes. Though repeated oxaliplatin administration had a systemic effect, we were unable to detect a pain-like behavioural phenotype in response to either cold or mechanical stimuli. Consequently, we cannot comment on whether the observed myeloid changes are associated with OIPN. Conclusions: Our discussion puts these results into the wider context of the field, advocating for greater transparency in reporting, alignment in experimental design and the introduction of more clinically relevant models. Only through joint concerted effort can we hope to increase our understanding of the underlying mechanisms of CIPN, including any immune contributions.

This PRISMA-Sc compliant scoping review project will seek to identify and report on any peer-reviewed, English-language journal articles published after 2010 that examined the association between the gut microbiome and/or immune system function on child neurodevelopment or behaviour up to 5 years of age. The research question for this scoping review is: What associations have been reported between the gut microbiome or immune system with continuous measures of development or behaviour within the first five years of life? We are limiting the search to peer-reviewed, English language journals published after 2010 to ensure relevance and interpretability. The final literature search for this review was completed in April, 2021. Data extraction will be completed by June 2021. Manuscript preparation will be completed before August 2021.

Pancreatic adenocarcinoma (PAAD) is a highly heterogeneous and immunosuppressive cancer. This study investigated the diversity of DNA damage repair (DDR) and immune microenvironment in PAAD by transcriptomic and genomic analysis. Patients with PAAD were divided into two DDR-based subtypes with distinct prognosis and molecular characteristics. The differential expression genes were mostly enriched in DDR and immune-related pathways. In order to distinguish high- and low-risk groups clinically, a DDR- and immune-based 5-gene prognostic signature (termed DPRS) was established. Patients in the high-risk group had inferior prognosis, a low level of immune checkpoint gene expression and low sensitivity to DDR-associated inhibitors. Furthermore, single-cell sequencing was used to observe the performance of the DDR-based signature in a high dimension, and immunohistochemistry was used to verify the relationship between the genes we identified and the prognosis of patients with PAAD. In conclusion, the DDR heterogeneity of PAAD was demonstrated, and a novel DDR- and immune-based risk-scoring model was constructed, which indicated the feasibility of DPRS in predicting prognosis and drug response in PAAD patients.

In mammals, β-defensins have been reported to play pivotal roles in sperm protection and fertilization. However, the function and mechanism of porcine β-defensin 129 (pBD129) in the sperm remain unclear. Here, we demonstrate that pBD129 is a glycosylated protein and broadly exists in accessory sex glands and coats the sperm surface. We inhibited the pBD129 protein on the sperm surface with an anti-pBD129 antibody and found that sperm motility was not significantly affected; however, sperm acrosome integrity and tyrosine phosphorylation levels increased significantly with time (p < 0.05) during capacitation. These changes were accompanied by an increase in sperm Ca2+ influx, resulting in a significantly reduced in vitro fertilization cleavage rate (p < 0.05). Further investigation revealed that treatment with recombinant pBD129 markedly restored the sperm motility in semen contaminated with Escherichia coli. The results suggest that pBD129 is not only associated with poor sperm motility after genital tract infection but can also protect the spermatozoa from premature capacitation, which may be beneficial for semen preservation.

The protocol registration for "Inflammation as an Emerging Comorbidity of Cerebral Palsy: A Scoping Review"

Background: Hepatitis C virus (HCV) therapy lowers risk of hepatocellular carcinoma (HCC). Little is known about factors driving/preceding HCC in treated persons. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) regulate host response and pathogenesis of disease. We investigated plasma levels of these RNAs and select serum markers before, during, and after HCV therapy, preceding HCC. Methods: Of 187 DAA treated HCV patients where therapy oriented longitudinal sampling was performed at a time without HCC diagnosis, 9 were subsequently diagnosed with HCC within 2 years of therapy. They were matched with 7 patients not diagnosed with HCC over the same time period. RNASeq was performed on plasma, and serum was assessed for biomarkers of inflammation by ELISA. Results: HCC diagnosis was 19 months (6–28) after therapy start in the HCC group. 73 and 63 miRs were differentially expressed at baseline (before DAA therapy) and 12 weeks after DAA therapy comparing HCC and non-HCC groups. Several lncRNA- showed differential expression as well. Several miRNA suppressors of cancer-related pathways, lncRNA- and mRNA-derived stabilized short RNAs were consistently absent in the plasma of patients who developed HCC. Serum IP10, and MCP-1 level was higher in the HCC group 12 weeks after therapy, and distinct miRNAs correlated with IP10 and MCP-1. Finally, in a focused analysis of 8 miRNAs best associated with HCC we observed expression of mi576 and mi-5189 correlation with expression of a select group of PBMC mRNA. Conclusions: These results are consistent with complex interplay between RNA-mediated host immune regulation and cancer suppression, strikingly skewed 12 weeks following therapy, prior to HCC diagnosis.

Prenatal alcohol exposure can disrupt the development of numerous systems, including the immune system. Indeed, alterations in cytokine levels may contribute to the neuropathological, behavioral, and cognitive problems, and other adverse outcomes observed in individuals with fetal alcohol spectrum disorders. Importantly, supplementation with the essential nutrient choline can improve performance in hippocampal-dependent behaviors; thus, the present study examined the effects of choline on plasma and hippocampal cytokines in adult rats exposed to ethanol in early development. From postnatal day (PD) 4–9 (third trimester equivalent), pups received ethanol (5.25 g/kg/day) or Sham intubations. Subjects were treated with choline chloride (100 mg/kg/day) or saline from PD10–30. On PD60, plasma and hippocampal tissue was collected before and after an immune challenge (lipopolysaccharide (LPS); 50 ug/kg). Prior to the immune challenge, ethanol-exposed subjects showed an overall increase in hippocampal pro-inflammatory cytokines, an effect mitigated by choline supplementation. In contrast, in the plasma, choline reduced LPS-related increases in pro-inflammatory markers, particularly in ethanol-exposed subjects. Thus, early choline supplementation may modify both brain and peripheral inflammation. These results suggest that early choline can mitigate some long-term effects of ethanol exposure on hippocampal inflammation, which may contribute to improved hippocampal function, and could also influence peripheral immune responses that may impact overall health.

Using pre-treatment gene expression, protein/phosphoprotein, and clinical data from the I-SPY2 neoadjuvant platform trial (NCT01042379), we create alternative breast cancer subtypes incorporating tumor biology beyond clinical hormone receptor (HR) and human epidermal growth factor receptor-2 (HER2) status to better predict drug responses. We assess the predictive performance of mechanism-of-action biomarkers from ∼990 patients treated with 10 regimens targeting diverse biology. We explore >11 subtyping schemas and identify treatment-subtype pairs maximizing the pathologic complete response (pCR) rate over the population. The best performing schemas incorporate Immune, DNA repair, and HER2/Luminal phenotypes. Subsequent treatment allocation increases the overall pCR rate to 63% from 51% using HR/HER2-based treatment selection. pCR gains from reclassification and improved patient selection are highest in HR+ subsets (>15%). As new treatments are introduced, the subtyping schema determines the minimum response needed to show efficacy. This data platform provides an unprecedented resource and supports the usage of response-based subtypes to guide future treatment prioritization.

The immune response is associated with the progression and prognosis of epithelial ovarian cancer (EOC). However, the roles of infiltrated immune cells and immune-related genes (IRGs) in EOC have not been reported comprehensively. In the current study, the differentially expressed genes (DEGs) were filtered based on the integrated gene expression data acquired from The University of California at Santa Cruz (UCSC) Genome Browser. Then, IRGs and transcriptional factors (TFs) were screened based on the ImmPort database and Cistrome database. A total of 501 differentially expressed IRGs, and 76 TFs were detected. A TF-mediated network was constructed by univariate Cox analysis to reveal the potential regulatory mechanisms of IRGs. Next, a nine immune-based prognostic risk model using nine IRGs (PI3, CXCL10, CXCL11, LCN6, CCL17, CCL25, MIF, CX3CR1, and CSPG5) was established. Based on the risk score worked out from the signature, the EOC patients could be classified into low-risk and high-risk groups. Furthermore, the immune landscapes, elevated by the cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm and the Tumor Immune Estimation Resource (TIMER) database, effectuated different patterns in two groups. Thus, an immune-based prognostic risk model of EOC elucidates the immune status in the tumor microenvironment, and hence, could be used for prognosis.

In this study, we developed a long noncoding RNA (lncRNA)‐based prognostic signature for stratification of patients with head a nd neck squamous cell carcinoma (HNSCC). In total, 493 HNSCC samples obtained from the Cancer Genome Atlas database were divided into training and testing cohorts (3:2 ratio). We identified 3913 immune‐related lncRNAs in the HNSCC training cohort by Pearson correlation analysis; only seven were independently associated with overall survival and were used to develop an immune‐related lncRNA prognostic signature (IRLPS) grouping of HNSCC patients into high‐ and low‐IRLPS subgroups. Univariate and multivariate Cox analyses revealed that low‐IRLPS patients had a better prognosis in all the cohorts, which was retained after stratification by sex, grade, and HPV status. Although the TNM stage was also an independent prognostic factor, the IRLPS had a better discriminability with higher AUC at the 3‐ and 5‐year follow‐ups in all cohorts. Low‐IRLPS samples had more immune cell infiltration and were enriched in immune‐related pathways, while high‐ IRLPS samples were enriched in metabolic pathways. A nomogram constructed including age, TNM stage, and IRLPS showed good calibration. Thus, IRLPS improves the prognostic prediction and also distinguishes different tumor microenvironment (TME) in HNSCC patients., We developed an immune‐related lncRNA signature for head and neck squamous cell carcinoma (HNSCC) patients, which could improve the prognostic prediction and distinguish different tumor microenvironments (TMEs) in HNSCC patients.

Colorectal cancer (CRC) is one of the most common malignant tumors, with the second-highest mortality of all 36 cancers worldwide. The roles of fatty acid metabolism in CRC were investigated to explore potential therapeutic strategies. The data files were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Univariate and least absolute shrinkage and selection operator (LASSO) Cox regression analyses were used to construct a prognostic risk score model with fatty acid metabolism-related genes for predicting prognosis in CRC. Patients with a high-risk score had a poorer prognosis in TCGA cohort than those with a low-risk score and were confirmed in the GEO cohort. Further analysis using the “pRRophetic” R package revealed that low-risk patients were more sensitive to 5-fluorouracil. A comprehensive evaluation of the association between prognostic risk score model and tumor microenvironment (TME) characteristics showed that high-risk patients were suitable for activating a type I/II interferon (IFN) response and inflammation-promoting function. Tumor Immune Dysfunction and Exclusion (TIDE) and SubMap algorithm results also demonstrated that high-risk patients are more suitable for anti-CTLA4 immunotherapy. Therefore, the evaluation of the fatty acid metabolism pattern promotes our comprehension of TME infiltration characteristics, thus guiding effective immunotherapy regimens., Graphical Abstract, A fatty acid prognostic risk score model is developed to enhance our comprehension of the role of fatty acid metabolism in CRC and guide more effective drug therapy strategies.

Background Breast cancer (BC), a very heterogeneous systemic disease, is the most frequently seen malignancy in women, especially in some developed countries or regions. Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki67 have been extensively used to predict the clinical outcome, but it is still a hotspot to search for more predictive prognostic markers. Ankyrin repeat and death domain containing 1A (ANKDD1A), which contains nine ankyrin repeats, has been discovered to play a role of tumor suppressor in glioblastoma multiforme (GBM). However, its role in BC remains unknown so far. Methods ANKDD1A expression and clinical information of BC were extracted from the TCGA dataset. Then, the ANKDD1A expression level was explored in BC from different perspectives, including clinical stage, molecular subtype, histology type and immune microenvironment. Afterwards, functional enrichment analysis of ANKDD1A co-expressed genes was carried out to estimate the role of ANKDD1A in BC, and the methylation status of ANKDD1A was evaluated by MEXPRESS. In addition, the correlation of ANKDD1A with immunocytes was explored, and survival analysis was carried out to evaluate the prognostic value of ANKDD1A in BC. Results ANKDD1A decreased in BC compared with the para-cancerous tissues. Additionally, ANKDD1A was up-regulated in early-stage BC, ER negative group, infiltrating lobular carcinoma, and the normal subtype in BC molecular subtypes. According to functional enrichment analysis, ANKDD1A co-expressed genes were mainly involved in the immune process. Also, our results revealed that ANKDD1A was tightly associated with T cells. Survival analysis suggested that, patients with higher ANKDD1A expression had more favorable prognosis than those with lower ANKDD1A expression. Conclusions ANKDD1A may serve as a critical gene in the pathogenesis of BC and the immune microenvironment of BC tissues.

The role of N7-methylguanosine(m7G)-related miRNAs in lung adenocarcinoma (LUAD) remains unclear. We used LUAD data from The Cancer Genome Atlas (TCGA) to establish a risk model based on the m7G-related miRNAs, and divided patients into high-risk or low-risk subgroups. A nomogram for predicting overall survival (OS) was then constructed based on the independent risk factors. In addition, we performed a functional enrichment analysis and defined the oxidative stress-related genes, immune landscape as well as a drug response profile in the high-risk and low-risk subgroups. This study incorporated 28 m7G-related miRNAs into the risk model. The data showed a significant difference in the OS between the high-risk and low-risk subgroups. The receiver operating characteristic curve (ROC) predicted that the area under the curve (AUC) of one-year, three-year and five-year OS was 0.781, 0.804 and 0.853, respectively. The C-index of the prognostic nomogram for predicting OS was 0.739. We then analyzed the oxidative stress-related genes and immune landscape in the high-risk and low-risk subgroups. The data demonstrated significant differences in the expression of albumin (ALB), estimated score, immune score, stromal score, immune cell infiltration and functions between the high-risk and low-risk subgroups. In addition, the drug response analysis showed that low-risk subgroups may be more sensitive to tyrosine kinase inhibitor (TKI) and histone deacetylase (HDAC) inhibitors. We successfully developed a novel risk model based on m7G-related miRNAs in this study. The model can predict clinical prognosis and guide therapeutic regimens in patients with LUAD. Our data also provided new insights into the molecular mechanisms of m7G in LUAD.

Background: The basement membrane (BM) serves as a major barrier to impede tumor cell invasion and extravasation during metastasis. However, the associations between BM-related genes and GC remain unclear. Methods: RNA expression data and corresponding clinical information of STAD samples were downloaded from the TCGA database. We identified BM-related subtypes and constructed a BM-related gene prognostic model using lasso-Cox regression analysis. We also investigated the single-cell properties of prognostic-related genes and the TME characteristic, TMB status, and chemotherapy response in high- and low-risk groups. Finally, we verified our results in the GEPIA database and human tissue specimens. Results: A six-gene lasso Cox regression model (APOD, CAPN6, GPC3, PDK4, SLC7A2, SVEP1) was developed. Activated CD4+ T cells and follicular T cells were shown to infiltrate more widely in the low-risk group. The low-risk group harbored significantly higher TMB and better prognosis, favoring immunotherapy. Conclusions: We constructed a six-gene BM-related prognostic model for predicting GC prognosis, immune cell infiltration, TMB status, and chemotherapy response. This research provides new ideas for developing more effective individualized treatment of GC patients.

We investigated the effects of dietary supplementation of lactic acid bacteria on the immune and antioxidant performance of weaned pigs. A total of 128 Duroc × Landrace × Yorkshire piglets weaned on day 28 with an average body weight of 8.95 ± 1.15 kg were selected and randomly divided into four treatment groups according to body weight and sex for a 28-day study. The four dietary treatments were basal diet (CON), and CON with 0.05% (LJ0.05), 0.1% (LJ0.1), and 0.2% (LJ0.2) Lactobacillus johnsonii RS-7, respectively. The lowest feed-to-gain ratio (F:G) was found when LJ0.1 was added to the diet. The addition of compound lactic acid bacteria to the diet increased the concentrations of TP, ALB, IgA, and IgM on day 14 and IgG, IgA, and IgM on day 28 (p < 0.05) in the blood, with trait values greater for pigs fed LJ0.1 than CON pigs (p < 0.05). Concentrations of antioxidants (CAT, T-AOC, MDA, T-SOD, and GSH) in serum, intestinal mucosa, spleen, liver, and pancreas improved. In summary, dietary supplementation of Lactobacillus johnsonii RS-7 improved the antioxidant and immune function of weaned piglets.

Chondrosarcoma (CHS) are heterogenous, but as a whole, represent the second most common primary malignant bone tumor entity. Although knowledge on tumor biology has grown exponentially during the past few decades, surgical resection remains the gold standard for the treatment of these tumors, while radiation and differentiated chemotherapy do not result in sufficient cancer control. An in-depth molecular characterization of CHS reveals significant differences compared to tumors of epithelial origin. Genetically, CHS are heterogenous, but there is no characteristic mutation defining CHS, and yet, IDH1 and IDH2 mutations are frequent. Hypovascularization, extracellular matrix composition of collagen, proteoglycans, and hyaluronan create a mechanical barrier for tumor suppressive immune cells. Comparatively low proliferation rates, MDR-1 expression and an acidic tumor microenvironment further limit therapeutic options in CHS. Future advances in CHS therapy depend on the further characterization of CHS, especially the tumor immune microenvironment, for improved and better targeted therapies., Cancers, 15 (9), ISSN:2072-6694

Background: The defense response is a type of self-protective response of the body that protects it from damage by pathogenic factors. Although these reactions make important contributions to the occurrence and development of tumors, the role they play in osteosarcoma (OS), particularly in the immune microenvironment, remains unpredictable. Methods: This study included the clinical information and transcriptomic data of 84 osteosarcoma samples and the microarray data of 12 mesenchymal stem cell samples and 84 osteosarcoma samples. We obtained 129 differentially expressed genes related to the defense response (DRGs) by taking the intersection of differentially expressed genes with genes involved in the defense response pathway, and prognostic genes were screened using univariate Cox regression. Least absolute shrinkage and selection operator (LASSO) penalized Cox regression and multivariate Cox regression were then used to establish a DRG prognostic signature (DGPS) via the stepwise method. DGPS performance was examined using independent prognostic analysis, survival curves, and receiver operating characteristic (ROC) curves. In addition, the molecular and immune mechanisms of adverse prognosis in high-risk populations identified by DGPS were elucidated. The results were well verified by experiments. Result: BNIP3, PTGIS, and ZYX were identified as the most important DRGs for OS progression (hazard ratios of 2.044, 1.485, and 0.189, respectively). DGPS demonstrated outstanding performance in the prediction of OS prognosis (area under the curve (AUC) values of 0.842 and 0.787 in the training and test sets, respectively, adj-p < 0.05 in the survival curve). DGPS also performed better than a recent clinical prognostic approach with an AUC value of only 0.674 [metastasis], which was certified in the subsequent experimental results. These three genes regulate several key biological processes, including immune receptor activity and T cell activation, and they also reduce the infiltration of some immune cells, such as B cells, CD8+ T cells, and macrophages. Encouragingly, we found that DGPS was associated with sensitivity to chemotherapeutic drugs including JNK Inhibitor VIII, TGX221, MP470, and SB52334. Finally, we verified the effect of BNIP3 on apoptosis, proliferation, and migration of osteosarcoma cells through experiments. Conclusions: This study elucidated the role and mechanism of BNIP3, PTGIS, and ZYX in OS progression and was well verified by the experimental results, enabling reliable prognostic means and treatment strategies to be proposed for OS patients.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovitis, joint damage and deformity. A newly described type of cell death, ferroptosis, has an important role in the pathogenesis of RA. However, the heterogeneity of ferroptosis and its association with the immune microenvironment in RA remain unknown. Synovial tissue samples from 154 RA patients and 32 healthy controls (HCs) were obtained from the Gene Expression Omnibus database. Twelve of twenty-six ferroptosis-related genes (FRGs) were differentially expressed between RA patients and HCs. Furthermore, the patterns of correlation among the FRGs were significantly different between the RA and HC groups. RA patients were classified into two distinct ferroptosis-related clusters, of which cluster 1 had a higher abundance of activated immune cells and a corresponding lower ferroptosis score. Enrichment analysis suggested that tumor necrosis factor-α signaling via nuclear factor-κB was upregulated in cluster 1. RA patients in cluster 1 responded better to anti-tumor necrosis factor (anti-TNF) therapy, which was verified by the GSE 198520 dataset. A diagnostic model to identify RA subtypes and immunity was constructed and verified, in which the area under the curve values in the training (70%) and validation (30%) cohorts were 0.849 and 0.810, respectively. This study demonstrated that there were two ferroptosis clusters in RA synovium that exhibited distinct immune profiles and ferroptosis sensitivity. Additionally, a gene scoring system was constructed to classify individual RA patients.

Deep learning (DL) can predict microsatellite instability (MSI) from routine histopathology slides of colorectal cancer (CRC). However, it is unclear whether DL can also predict other biomarkers with high performance and whether DL predictions generalize to external patient populations. Here, we acquire CRC tissue samples from two large multi-centric studies. We systematically compare six different state-of-the-art DL architectures to predict biomarkers from pathology slides, including MSI and mutations in BRAF, KRAS, NRAS, and PIK3CA. Using a large external validation cohort to provide a realistic evaluation setting, we show that models using self-supervised, attention-based multiple-instance learning consistently outperform previous approaches while offering explainable visualizations of the indicative regions and morphologies. While the prediction of MSI and BRAF mutations reaches a clinical-grade performance, mutation prediction of PIK3CA, KRAS, and NRAS was clinically insufficient.

This investigation explored the impact of dietary frankincense resin oil (FO) on growth performance parameters, intestinal histomorphology, fatty acid composition of the breast muscle, and the immune status of broilers. We allotted 400, three-day-old, male chicks (Ross 308 broiler) into four treatment groups (ten replicates/group; ten chicks/replicate). They were fed a basal diet with different concentrations of FO (0, 200, 400, and 600 mg kg−1). FO supplementation increased the overall body weight (BW) and body weight gain (BWG) by different amounts, linearly improving the feed conversion ratio with the in-supplementation level. Total feed intake (TFI) was not affected. Growth hormones and total serum protein levels also linearly increased with the FO level, while albumin was elevated in the FO600 group. Moreover, total globulins increased linearly in FO400 and FO600 treatment groups. Thyroxin hormone (T3 and T4) levels increased in all FO treatment groups without affecting glucose and leptin serum values. Different concentrations of FO supplementation in the diet increased the activities of Complement 3, lysozyme, and interleukin 10 levels in the serum. Dietary FO in broilers increased the total percentage of n-3 and n-6 fatty acids. It also increased the ratio of n-3 to n-6 linearly and quadratically. Additionally, FO supplementation led to the upregulation of immune clusters of differentiation 3 and 20 (CD3 and CD20) in the spleen, along with improving most of the morphometric measures of the small intestine. In conclusion, FO up to 600 mg kg−1 as a feed additive in broiler chicken production is valuable for promoting their growth, intestinal histomorphology, and immune status along with enriching breast muscle with polyunsaturated fatty acids (PUFA).

Pancreatic cancer has poor survival despite modern-day advances in its management. At present, there are no available biomarkers that can predict chemotherapy response or help inform prognosis. In more recent years, there has been increased interest in potential inflammatory biomarkers, with studies revealing a worse prognosis of patients with a higher neutrophil-to-lymphocyte ratio in a range of tumour types. Our aim was to assess the role of three inflammatory biomarkers in peripheral blood in predicting chemotherapy response in patients with earlier disease treated with neoadjuvant chemotherapy and as a prognostic marker in all patients that underwent surgery for pancreatic cancer. Using retrospective records, we discovered that patients with a higher neutrophil-to-lymphocyte ratio (>5) at the time of diagnosis had worse median overall survival than those with ratios ≤5 at 13 and 32.4 months (p = 0.001, HR 2.43), respectively. We were able to appreciate a correlation between a higher platelet-to-lymphocyte ratio and increased residual tumour in the histopathological specimen in patients receiving neoadjuvant chemotherapy; however, the association was weak (p = 0.03, coefficient 0.21). Due to the dynamic relationship between the immune system and pancreatic cancer, it is unsurprising that immune markers may be useful as potential biomarkers; however, larger prospective studies are needed to validate these findings.

Guang-zhao Huang,1,* Qing-qing Wu,1,* Ze-nan Zheng,1,* Ting-ru Shao,1 Fei Li,1 Xin-yan Lu,1 Heng-yu Ye,1 Gao-xiang Chen,1 Yu-xing Song,1 Wei-sen Zeng,2 Yi-long Ai,3 Xiao-zhi Lv1 1Department of Oral & Maxillofacial Surgery, NanFang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People’s Republic of China; 2Department of Cell Biology, School of Basic Medical Science, Southern Medical University, Guangzhou, Guangdong Province, People’s Republic of China; 3Foshan Stomatological Hospital, School of Stomatology and Medicine, Foshan University, Foshan, Guangdong Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiao-zhi LvDepartment of Oral & Maxillofacial Surgery, NanFang Hospital, Southern Medical University, Baiyun District, Guangzhou, Guangdong Province, 510080, People’s Republic of ChinaTel +86 13724811658Email lxzsurgeon@126.comYi-long AiFoshan Stomatological Hospital, School of Stomatology and Medicine, Foshan University, Chancheng District, Foshan, Guangdong Province, 528000, People’s Republic of ChinaTel +86 13827755774Email aiyilong@126.comPurpose: Plenty of studies showed that the immune system was associated with cancer initiation and progression. This study aimed to explore the prognostic biomarkers from immune-related genes (IRGs) in oral squamous cell carcinoma (OSCC).Materials and Methods: RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) and IRGs and transcription factors (TFs) were extracted. Then, the co-expression network between IRGs and TFs was constructed using the “WGCNA” package in R software. Furthermore, a gene expression signature according to IRGs was constructed to predict OSCC prognosis and its accuracy was validated by survival analysis. Subsequently, correlation analyses between risk-score and immune cells level and clinical parameters were performed. Finally, immune-related biomarkers were selected and further investigated using gain-of-function assays in vitro.Results: A total of 32 normal cases and 317 OSCC cases were selected in our study. Differentially-expressed analysis indicated that there were 381 differentially-expressed IRGs and 62 TFs in OSCC. Among them, 25 TFs and 21 IRGs were enrolled in the co-expression network. Furthermore, we found that gene expression signature on the basis of 10 IRGs could predict the prognosis accurately and a high-risk score based on gene expression signature meant a high T classification, terminal clinical stage, and low immune cells level in OSCC. Finally, cathepsin G (CTSG) was identified as a potential immune-related biomarker and therapeutic target in OSCC.Conclusion: In conclusion, IRGs were directly involved in the development and progression of OSCC. Furthermore, CTSG was identified as a potential independent biomarker and might be an immunotherapeutic target in OSCC treatment.Keywords: oral squamous cell carcinoma, immune, biomarker, bioinformatics, CTSG

Introduction:This study is an experimental setup to examine the effects of splenectomy, applied to multi-trauma patients who are commonly seen in emergency traumatology practices and on fracture healing processes with respect to histopathological, biomechanical, and radiological aspects.Methods:Further, 32 male Sprague Dawley rats (10 months old; average weight 394.5±28.3 g) are included in the study. In a dark environment, rats are fed at 22 °C for 12 hours with standard rodent food ad libitum. They are divided into two groups: splenectomy (total splenectomy and long bone fracture healing) and control (only long bone fracture healing n=16). Four months after surgery, rats are killed, and callus tissues in fractured femurs were examined histopathologically, radiologically, and biomechanically.Results:In the radiological analysis of the femur materials in relation to the case and control groups, there was no substantial difference in the Goldberg classification score (p˃0.05). Similarly, no substantial difference was observed (p˃0.05) in histopathological examinations and biomechanical analysis conducted in femur samples.Conclusion:When all of the results obtained in our study are evaluated together, it was concluded in the rats to which splenectomy is applied that this did not affect fracture healing histopathologically, biomechanically, and radiologically, despite the modification of immune modulators.